This program is devoted to the characterization of metalloenzyme active sites through the use of multinuclear, multifrequency (9; 35; 95 GHz) CW and pulsed electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies. The core of our research will be the study of oxygen-activating, Diron centers of, Hemes of, Methane Monoxygenase Nitric Oxide Synthase Ribonucleotide Reductase Cytochromes P450 This effort will emphasize the characterization of key catalytic intermediates. The uses of ENDOR/ESEEM will be expanded to the investigation of diamagnetic enzyme states, by making them paramagnetic through low-temperature reduction. Advances in resolution and sensitivity with our high-frequency pulsed ENDOR spectrometers will extend the reach of ENDOR beyond the primary metal ligands; substrate recognition will be analyzed through the determination of precise metrical structures for enzyme-bound substrates/inhibitors. These studies are linked by a desire to understand (i) the processes by which iron centers react with 02 to generate reactive high-valent [Fen- Om] species, (ii) the nature of these reactive species, and (iii) the means by which enzymes control their reaction with substrate.